Genista tridentata Phytochemical Characterization and Biological Activities: A Systematic Review

Simple Summary This study systematically reviewed the literature on the bioactivities and phytochemical profile of Genista tridentata (L.) Willk, which is an edible plant used in folk medicine. Four databases (PubMed, GoogleScholar, Repositórios Cientificos de Acesso Aberto de Portugal (RCAAP), and ScienceDirect) were searched from inception up to 31 December 2022. Among 34 eligible papers, the flowers and aerial parts were extensively studied, with aqueous extracts being the most commonly used. The findings suggest that Genista tridentata has high potential as a natural source of antioxidants and preservatives for the food/cosmetic industry, offering health benefits. Additionally, its year-round collectability provides an advantage for industrial applications. Abstract Genista tridentata (L.) Willk., known as “prickled broom”, is a Leguminosae (Fabaceae) species native to the Iberian Peninsula, Morocco, Algeria, and Tunisia. It is used in folk medicine as an anti-inflammatory, for gastrointestinal and respiratory disorders, rheumatism, and headaches, to lower blood pressure, against hypercholesterolemia and hyperglycemia. This study aimed to systematically review the literature on the bioactivities and phytochemical profile of Genista tridentata to understand its pharmacological potential. For this, four electronic databases (PubMed, GoogleScholar, Repositórios Cientificos de Acesso Aberto de Portugal (RCCAP), and ScienceDirect) were searched from inception up to 31 December 2022. From a total of 264 potentially eligible studies considered for screening, 34 papers were considered eligible for this systematic review. The sampling included 71 extracts, collected mainly in Portugal. Genista tridentata extracts present a high level of flavonoids and phenolic compounds. The flowers and aerial parts of the plant were the most studied, and aqueous extracts were the most used. The results predict a high potential for the application of Genista tridentata as a new source of natural antioxidants and preservatives for the food industry with subsequent health benefits, such as the production of nutraceuticals. Moreover, the results indicate that the plant can be collected at all seasons of the year, which represents a benefit for the industry.


Introduction
Wild edible plants are an important piece of the cultural and genetic heritage of distinct world regions, representing high importance, predominantly in rural and suburban areas [1].Furthermore, they are interesting sources of bioactive compounds and need recognition as considerable contributors to human health promotion and disease prevention [2].

Literature Search
A schematic representation of the literature screening process is provided in Figure 1.In the first triage, after the removal of the duplicates, 2001 results were identified.From these, 719 were excluded because of the language criteria, then 1282 were excluded as their topic was outside the scope of this review.The remaining 264 articles were screened by title and abstract, with 230 being excluded for the following reasons: 88 because of document type (review articles and grey literature), 79 did not provide data concerning biological or pharmacological activities; 1 was excluded because it reported mixed results (G.tridentata extract was mixed with other plant species); 13 were excluded because they reported results of isolated compounds not directly related to G. tridentata and, 14 were excluded because of missing data.Thus, 34 articles were included in the systematic review.
The reports included identification and analyses of the phytochemical compounds present in G. tridentata extracts and their bioactivities.

Phytochemical Characterization
The main compounds found are flavonoids, as well as hydroxycinnamic acids and hydroxybenzoic acids (Table 1, Figure 2).Additionally, extracts collected in the flowering period (May), as well as flowers, presented a more diverse phytochemical profile than extracts collected during the rest of the year.
Other classes of compounds found in the literature include essential oils (EOs) (Table 2).Grosso et al. (2007) [11] studied the EOs isolated by hydrodistillation and distillationextraction of flowers, stems and leaves, and aerial parts of different populations.Another study [29] also characterized EOs of G. tridentata using the same methodology.The results show that G. tridentata samples presented a yellowish oil with a yield of <0.05% (v/w).The dominant components of the oils were phenylpropanoids, more abundant in aerial parts, and oxygen-containing monoterpenes in the flowers, stems, and leaves.Additionally, Faria et al. (2013Faria et al. ( , 2016) ) [31,33] reported cis-theaspirane and trans-theaspirane as the main components.

Phytochemical Characterization
The main compounds found are flavonoids, as well as hydroxycinnamic acids and hydroxybenzoic acids (Table 1, Figure 2).Additionally, extracts collected in the flowering period (May), as well as flowers, presented a more diverse phytochemical profile than extracts collected during the rest of the year.Other classes of compounds found in the literature include essential oils (EOs) (Table 2).Grosso et al. ( 2007) [11] studied the EOs isolated by hydrodistillation and distillation-extraction of flowers, stems and leaves, and aerial parts of different populations.Another study [29] also characterized EOs of G. tridentata using the same methodology.The results show that G. tridentata samples presented a yellowish oil with a yield of <0.05% (v/w).The dominant components of the oils were phenylpropanoids, more abundant in aerial parts, and oxygen-containing monoterpenes in the flowers, stems, and leaves.Additionally, Faria et al. (2013, 2016) [31,33] reported cis-theaspirane and trans-theaspirane as the main components.
Vitor et al. ( 2004) [9] demonstrated that 3-O-glucoside isoquercitrin and prunetin, isolated from an aqueous extract of G. tridentata, were shown to prevent endothelial oxidative damage and have radical scavenging activity inhibiting O 2− generation.
Moreover, in two studies [19,35], aqueous extracts of G. tridentata were assayed for their capacity to protect deoxyribose from degradation.Results showed that the strongest antioxidant effect was associated with hot water extraction, compared to cold extraction.[14] assessed the anti-inflammatory activity of methanolic, petroleum ether, and ethyl acetate extracts of G. tridentata using the TNF-α inhibition assay in human monocytes.The results indicated an inhibition level between 80 and 60% for all extracts.Also, more recently, Simões et al. (2020) [4] evaluated the anti-inflammatory activity of G. tridentata ethanolic extracts (roots, stems, and leaves) through the inhibition of LPS-NO (lipopolysaccharide-nitric oxid) production and demonstrated a downregulation of the Nos2 gene.The other two studies also demonstrated a decrease in the transcription of the pro-inflammatory genes IL1b, IL6, and Ptgs2 with stems and root extracts.LPS-NO production was decreased in flower-based G. tridentata aqueous extract [15,34] and in ethanolic extract [15].Mota et al. (2022) [40] evaluated the myeloperoxidase (MPO) inhibition by ethanolic extract of G. tridentata and demonstrated an IC 50 of 0.032 ± 0.004 mg/mL.

Antifungal and Antibacterial Activity
The results of the antifungal activity of G. tridentata are presented in Table 4.The results showed that G. tridentata extracts are effective against six Candida strains, namely C. albicans, C. glabrata, and C. parapsilosis species, while the majority of C. tropicalis strains were resistant [37].The antifungal activity of aqueous extract of G. tridentata flowers was also demonstrated against Aspergillus niger, Aspergillus versicolor, Penicillium funiculosum, Penicillium aurantriogriseum and Penicillium verrucosum [15,34].
Table 4 also shows the results for the screening of the antibacterial activity of G. tridentata against S. aureus and E. coli, Salmonella Typhimurium, Bacillus cereus, and Listeria monocytogenes.G. tridentata extracts display moderate to strong antibacterial activity against S. aureus [17].

Cytotoxicity
The cytotoxicity of G. tridentata aqueous extract was assessed using a human colorectal epithelial adenocarcinoma cell line (Caco-2) and a human cervical adenocarcinoma cell line (HeLa) [20].Additionally, the cytotoxicity of G. tridentata flower aqueous extract was also assessed using a human hepatocarcinoma cell line (HePG2) [6].The results showed no toxicity in the cell lines evaluated.
Ferreira et al. ( 2012) [6] also evaluated the toxicological effects of G. tridentata flower aqueous extract in mitochondrial respiratory rates (state 4 and state 3 respiration and FCCP-stimulated respiration) and respiratory indexes (respiratory control ratio and P/O ratios) in rat liver mitochondria.The results demonstrated no toxicity of the extracts since no identifiable interactions with respiratory enzymes were detected.

Quality Assessment
The quality assessment of the works was performed using the ARRIVE quality guidelines, with each criterion being rated with a score between 0 and 1 (0-Absent; 0.5-Incomplete or not applicable; 1-Complete).About 56.25% of the studies were considered good quality, scoring higher than 15 out of 20, and 43.75% were considered moderate quality, rating between 12 and 15 out of 20.The mean score was determined for each work, and the total mean score for the quality of included works was 15.18 ± 1.14 (Table 5).

Phytochemical Characterization
The phytochemical profile (Table 1) clearly demonstrates that extract composition varies considerably with the extraction method, probably due to the solubility of the compounds.Methanolic and ethanolic solvents are less polar in nature, disrupting cell walls (nonpolar structures) and triggering the release of phenolic compounds; while water presents a high polarity index, it is best to extract compounds with higher polarity [35].As the extraction solvent influences the plant extract composition [37,39], reports should carefully describe the extraction method to facilitate comparison between laboratories.The location and the vegetative stage also influence the phytochemical profile of extracts; consequently, it is important to be careful in the analyses of data from the works that do not indicate the sampling period.Plant extracts collected in the flowering period (May), as well as flowers, displayed a more diverse phytochemical profile than extracts collected during the rest of the year.
Furthermore, the in vitro extracts exhibited a different composition content than wild extracts [24].These variations could be explained because some compounds are not essential in in vitro plants as a defense mechanism against harmful environmental conditions, and consequently, they do not produce them [24].
Moreover, works performing a phytochemical characterization of G. tridentata are scarce, especially when using other extraction solvents, such as acetone.Another important parameter is the quantification of the phytochemical compounds identified, often overlooked by researchers.
Regarding EOs (Table 2), the variability between samples from the same location and year indicates that the chemical composition of G. tridentata oils is not a consequence of climatic factors in distinct years but a consequence of other intrinsic factors related to the plant (ratio plant/organ, vegetative state, genetics) and its interaction with the environment (type of the soil, climate, harvest time during the day) [44].

Plant Bioactive Compounds as Antioxidants
There is no general procedure that can determine the antioxidant capacity of all samples precisely and quantitatively [45].Standardized approaches to evaluate antioxidant activity should follow specific requirements, and consequently, the procedures fall into two broad categories: radical scavenging activity and lipid peroxidation inhibition [46].
The variability of assays facilitates the establishment of G. tridentata potential [3].However, it is impossible to compare between essays since there is no standardization of procedures, and G. tridentata extracts are obtained with different solvents and from different locations.Additionally, not all evaluated works performed replicates of the antioxidant tests [5,12,18,24,27,41], so they should be repeated.
Furthermore, the anti-oxidative effects of plant extracts on deoxyribose and DNA were promising concerning practical applications of G. tridentata as an ingredient in the formulation of nutraceutical beverages and foods, as well as cosmetic formulations, since they add a protective effect.
The phytochemical characterization (Tables 1 and 3) of G. tridentata extracts demonstrates a high level of phenolic compounds, especially flavonoids.Structurally, phenolic compounds possess an aromatic ring with one or more hydroxyl substitutes.The aromatic feature and highly conjugated system with multiple hydroxyl groups make these compounds good electron/hydrogen atom donors, neutralizing free radicals and other ROS [47].
The antioxidant activity of phenolics can be based on hydrogen atom transfer and/or single-electron transfer.Nevertheless, the antioxidant potential of a phenolic compound depends on the number and the position of hydroxyl groups in the molecule [47,48].
Likewise, the degree of hydroxylation also influences the antioxidant activity.The longer distance separating the carbonyl group and the aromatic ring appears to improve the antioxidant capability.Additionally, a greater number of hydroxyl aromatic rings, such as in flavonoids, increased the antioxidant activity [47].
Importantly, flavonoids and their glycosylated derivates in G. tridentata extracts were stable during in vitro digestion, which indicates a strong antioxidant activity [20].Nevertheless, this analysis should be repeated since this single work evaluated gastric and pancreatic digestion, and their quality assessment indicates some deficiencies (Table 5).
Some studies showed a significant synergistic effect between bioactive compounds and, consequently, an increase in antioxidant activity [49,50].Therefore, the high antioxidant capacity of G. tridentata extracts is probably due to a synergistic interaction between their phytochemical components rather than due to a single chemical element.

Anti-Inflammatory Activity
Several health benefits attributed in traditional medicine to G. tridentata infusions or decoctions are linked to their anti-inflammatory activity [5,51].Nevertheless, scientific reports focused on the anti-inflammatory potential of G. tridentata extracts are scarce and do not identify the mechanism of action.The phytochemical characterization of G. tridentata extracts demonstrated their richness in flavonoids, frequently correlated with anti-inflammatory activity [3,52].Additionally, numerous studies demonstrated the anti-inflammatory potential of biochanin A [53][54][55][56], prunetin [57,58], genistein [59,60], rutin [61,62] and taxifolin [63,64] in decreasing inflammatory mediators as TNF-α, inhibiting the production of pro-inflammatory cytokines (IL-1β, IL-6, IL-33) and of proinflammatory enzymes as nitric oxide synthases.Pinto et al. (2020) [3] provided a comprehensive review that explores the potential mechanisms of action of several bioactive compounds also found in G. tridentata concerning their anti-inflammatory activity.
However, it would be important to evaluate the synergistic effects of the bioactive compounds, as well as the evaluation of extracts obtained with different solvents.Importantly, the in vivo evaluation of G. tridentata extracts could be central to understanding the mechanisms of action and establishing the pharmacological effects of the plant's extracts.

Antifungal and Antibacterial Activity
The antifungal activity of G. tridentata against Candida strains is probably associated with the existence of the flavonoids, quercetin, and genistein derivates.Quercetin inhibits growth [65,66] and prevents microbial biofilm formation, possibly by reducing the cellular adhesion to abiotic surfaces, as described for C. albicans [67].Also, genistein derivates inhibit C. albicans colony formation [68,69].
The antibacterial potential of G. tridentata can result from the higher content of phenolic compounds, flavonols, and isoflavones, which improves the antimicrobial power of extracts.
Additionally, the presence of taxifolin, genistin, and biochanin was previously reported as having an antibacterial activity [70][71][72], reducing bacteria resistance mechanisms and conducting delayed protein synthesis in S. aureus involved in the production of enzymes and nucleic acids required for bacterial growth [17,71,73].As a result, membrane permeability increases, leading to a decline in bacteria survival.Also, flavonoids display a strong capacity to form complexes with bacteria cell walls, inhibiting bacteria growth [73][74][75].
Identically, rutin, isoquercitrin, and quercetin can act as antimicrobial agents, inhibiting nucleic acid and cytoplasmic membrane synthesis as well as bacterial metabolism [73].Importantly, the above-mentioned phenolic compounds are more effective against Grampositive bacteria [76], like S. aureus.Consequently, these compounds could have a bacteriostatic effect instead of a bactericidal action [17].
Regardless of the need for additional studies to clarify the mechanisms of action, it is possible to conclude that G. tridentata can be valuable in treating or complementing commercial drugs due to its bacteriostatic and antifungal potential.However, the extraction solvent influences the plant extracts bioactivity [37,39].Thus, based on that, it could be important to evaluate the antifungal and antibacterial effects of other extraction methods of G. tridentata.

Cytotoxicity
Firstly, it is important to clarify that human cell lines are preferred to primary cultures from animals, as they decrease species-related variations that can occur during the extrapolation of the results [77].Secondly, data obtained by Serralheiro et al. (2013) [20], Ferreira et al. (2012) [6], and Caleja et al. (2019) [34] demonstrated that G. tridentata aqueous extracts could be appropriately used in traditional medicine, in cooking and the preservation of aliments against oxidative stress.

Nematocidal/Nematotoxicity Activity
Although several studies showed that EOs display high nematocidal activities [78,79], literature about G. tridentata EOs is still limited.The mechanism of action of EOs is complex and occurs through different pathways, and some authors suggest that the interference of EOs in the nematode nervous system [80] is related to the neuromodulator octopamine [81] or GABA-gated chloride channels in insects [82].Moreover, as in bacteria or fungi, EOs might disturb the cell membrane of the nematode by altering its permeability [80].
Importantly, the phytochemical profile of G. tridentata EOs (Table 2) demonstrated the presence of carvacrol and geraniol.Some studies demonstrated the nematocidal activity of carvacrol, facilitated via tyramine receptor, that triggered a signaling cascade and, by interacting with a receptor-like SER-2, led to nematode mortality [83].Similarly, the activity of geraniol in membrane disturbing, changing membrane-bound protein, and the intracellular signaling pathways [84,85] has also been described.Although experimentally, EOs of G. tridentata display nematocidal activity; it is important to complement the in vitro tests with in vivo soil-based trials to validate the efficiency of this plant EOs.
The main advantages of the nematocidal/nematotoxicity activity of EOs are their minimal toxicity to mammalians and their low environmental persistence, making them safe and compatible as biological control agents [86].

Other Activities
Results obtained by Paulo et al. (2008) [27] with the isolated compounds isoquercitrin and sissotrin revealed a time-dependent antihyperglycemic activity, indicating the presence of bioactive compounds that differently influence glucose uptake.While these results discourage the use of the plant in the control of glycemic blood levels, they highlight a possible post-prandial hypoglycemic effect of isoquercitrin.However, this work [27] presents several imperfections, especially regarding scientific work with animals, not revealing the number of animals used or blinding techniques.
Nevertheless, performing more studies to assess the antihyperglycemic activity of G. tridentata extracts is important to understand the mechanism of action of its bioactive compounds.Serralheiro et al. (2013) [20] demonstrated using a colorimetric assay that the G. tridentata aqueous extracts display anti-acetylcholinesterase activity.Acetylcholine (ACh) is an important excitatory neurotransmitter responsible for peristaltic movements [87,88] and is also responsible for the regulation and establishment of an adequate environment for enzymatic digestion and absorption and the lubrication of intestinal material [89].Serralheiro et al. (2013) [20] suggested that the flavonoids present in the extract may play an essential role in the inhibition of ACh by interacting with their active bind sites.Falé et al. (2012) [90] also showed small alterations in the ACh, namely in the aromatic amino acids, that affect enzymatic activity.The alterations occur in the presence of quercetin, romarinic acid, luteolin, and apigenin [90], all of which are also present in G. tridentata aqueous extracts.Falé et al. (2014) [22] intended to understand the mechanism of action of G. tridentata extract in cholesterol in an in vivo experiment and found a reduction of 22% of plasma total cholesterol.Apart from the limited number of volunteers, the authors also do not refer to the control of other important variables such as diet and physical exercise.They also performed in vitro studies to evaluate the Inhibitory concentration of HMG-CoA of G. tridentata extract HMG-CoA reductase.The downregulation of HMGCR is directly associated with cholesterol reduction via SREBP-2 activation as it acts in the upregulation of low-density lipoprotein receptor (LDLR) that increases the exclusion of cholesterol-rich low-density lipoprotein (LDL) particles from the blood circulation [91,92].
The same authors [22] also performed permeation studies in Caco-2 monolayer cells.The results demonstrated the bioavailability of some bioactive compounds, suggesting the biochanin A glucoside as a specific transporter of glycoside moiety that may be involved in the reduction of cholesterol [22].Sadri et al. (2017) [93] also demonstrated that oral administration of biochanin A (10 and 15 mg/kg) in rats significantly decreased serum triglycerides, total cholesterol, and LDL cholesterol.

Conclusions
Genista tridentata extracts present a high extraction yield and have been described to possess high levels of flavonoids and phenolic compounds, suggesting a high potential for its application as a new source of natural antioxidants and preservatives for the food industry or in products with health benefits, such as nutraceuticals.Moreover, the results indicate that the plants can be collected at all seasons of the year, which represents an added benefit for the industry.

Figure 1 .
Figure 1.Schematic representation of the PRISMA flow chart depicting the literature screening process.

Figure 1 .
Figure 1.Schematic representation of the PRISMA flow chart depicting the literature screening process.

Figure 2 .
Figure 2. Classification of phytochemical compounds identified in Genista tridentata.Each distinct category is associated with a unique color.

Figure 2 .
Figure 2. Classification of phytochemical compounds identified in Genista tridentata.Each distinct category is associated with a unique color.

Table 1 .
Major biologically active compounds were found in several samples of G. tridentata (X-detected; NA-not available; ND-not detected).The table's color scheme was aligned with Figure2.

Table 2 .
Composition (%) of the essential oils of G. tridentata isolated by hydrodistillation, collected in different years and locations.

Table 3 .
Antioxidant activity and phenolic and flavonoid content in several samples of G. tridentata.(AA-ascorbic acid; AAE-ascorbic acid equivalent; ext-extract; GAE-gallic acid equivalent; ip-inhibition percentage; CAE-caffeic acid equivalent; ClAE-chlorogenic acid equivalent; inf-infusion; Pt-Portugal; Sp-Spain; T-Trolox.TAC-total antioxidant capacity; in cases where the units are not indicated, the unit indicated in the first line must be considered; NA-not available; NR-not reported).